Discovery of new Hsp90–Cdc37 protein–protein interaction inhibitors: in silico screening and optimization of anticancer activity

The interaction between heat shock protein 90 (Hsp90) and Hsp90 co-chaperone cell-division cycle 37 (Cdc37) is crucial for the folding and maturation of several oncogenic proteins, particularly protein kinases. This makes the inhibition of this protein–protein interaction (PPI) an interesting target for developing new anticancer compounds. However, due to the large interaction surface, developing PPI inhibitors is challenging. In this work, we describe the discovery of new Hsp90–Cdc37 PPI inhibitors using a ligand-based virtual screening approach. Initial hit compounds showed Hsp90 binding, resulting in anticancer activity in the MCF-7 breast cancer cell line. To optimize their antiproliferative effect, 35 analogs were prepared. Binding affinity for Hsp90 was determined for the most promising compounds, 8c (Kd = 70.8 μM) and 13g (Kd = 73.3 μM), both of which interfered with the binding of Cdc37 to Hsp90. This resulted in anticancer activity against Ewing sarcoma (SK-N-MC), breast cancer (MCF-7), and leukemia (THP-1) cell lines in vitro. Furthermore, compounds 8c and 13g demonstrated the ability to induce apoptosis in the Ewing sarcoma cell line and caused a decrease in the levels of several known Hsp90 client proteins in MCF-7 cells, all without inducing the heat shock response.


Introduction
Proteins are among the key cellular building blocks, as their roles include structural support, immune function and signal transduction. 13][4] Therefore, correct folding and stabilization of oncoproteins is necessary in cancer pathology and the role of chaperones in the development of malignancies is clear. 5,6Perhaps one of the most studied cancer-related chaperones is heat shock protein 90 (Hsp90), 7,8 which is essential for the correct folding of more than 400 client proteins.[10] The inhibition of the chaperone cycle of Hsp90 can lead to the downregulation of oncogenic drivers, emphasizing the potential of Hsp90 inhibitors as anticancer agents.The overexpression of Hsp90 and its higher affinity for ATP in cancer cells provide a basis for selective targeting of malignantly transformed cells. 10,11Unfortunately, the rst discovered Hsp90 inhibitor, geldanamycin, 12 was too unstable and potentially toxic for clinical use. 13,14However, optimized analogs like 17-AAG and 17-DMAG have entered clinical trials.Subsequently, other structural classes with similar mechanisms of action were proposed as clinical candidates.4][15] Despite potent on-target activity, these inhibitors were not as successful in the clinic.It was not until 2022 that the rst N-terminal Hsp90 inhibitor, pimitespib, was approved for the treatment of gastrointestinal stromal tumors in Japan. 16,17ue to limited clinical success of the rst Hsp90 N-terminal inhibitors, other approaches targeting various structural and functional traits of Hsp90 started to emerge. 13The Hsp90 chaperone family is highly conserved: a charged linker connects the ATP-binding NTD to the client-binding middle domain (MD), which then extends into the C-terminal domain (CTD), where dimerization occurs. 18Moreover, all three domains of Hsp90 participate in protein-protein interactions (PPI) with different co-chaperones essential for normal progression through chaperone cycle stages. 19,20For instance, activator of ATPase homologue 1 (Aha1), which binds to the MD and NTD, enhances the otherwise low ATPase activity of Hsp90. 21,22onversely, the NTD-binding p23 inhibits ATPase activity and prolongs Hsp90's interaction with client proteins. 23,24p23 acts at a later stage of the chaperone cycle, 25 while the Hsp70/Hsp90 organizing protein (Hop) binds the CTD of Hsp90 at the beginning, recruiting client proteins to Hsp90.Clients like glucocorticoid receptors are transferred from a complex with Hsp70 to Hsp90 by Hop. 26,27On the other hand, protein kinases and some other client proteins rely on cell division cycle 37 (Cdc37/p50) for their recruitment to Hsp90.Like Hsp90, the expression levels of Cdc37 are increased in cancer cells, 28 making Hsp90-Cdc37 PPI particularly intriguing for the design of potential cancer growth inhibitors.9][30][31][32] Disruption of Hsp90-Cdc37 PPI could result in a more selective approach to disrupting the Hsp90 chaperone cycle.
Despite the challenging druggability of PPIs, several classes of Hsp90-Cdc37 PPI inhibitors have been described to date (Fig. 1). 33,346][37][38][39][40][41][42][43] This changed in 2018 and 2019 with the discovery of DCZ311244 44 and DDO-5936. 45Wang and colleagues found that the binding of DDO-5936 is largely dependent on Asp47 on the NTD of Hsp90.They also identied Arg167 and Gln133 as indispensable for the binding of Cdc37 to Hsp90. 45Subsequently, they prepared a library of analogs to establish the structure-activity relationships (SAR) of this compound class. 46,47The binding site of DDO-5936 on Hsp90 NTD was established through mutagenesis, protein NMR studies, and binding affinity measurements with both wild-type and mutated Hsp90.These assays identied Asp47 as a key binding determinant, as DDO-5936 showed no affinity for the Asp47Ala mutant of Hsp90 (K d > 100 mM for the mutant vs.K d = 3.86 mM for the wild-type).This suggests that DDO-5936 binds to a previously unknown NTD binding site adjacent to the ATPbinding site. 45However, recent cryo-EM structures of the fulllength Hsp90b dimer in complex with Cdc37 and various client proteins 29,31,48 indicate that interaction with Asp42 (Hsp90b amino acid numbering) might not be possible in a cellular context, as the Leu388-Met394 loop folds into the proposed binding site, possibly rendering it inaccessible.
Based on these discoveries, we decided to investigate whether our enhanced understanding of the Hsp90 N-terminal binding site 45 and its inhibitors 46,47 could be combined with computer-aided drug design to develop new small-molecule inhibitors of Hsp90-Cdc37 PPI.In this study, we employed ligand-based molecular modeling approach to identify hit compounds capable of inhibiting Hsp90-Cdc37 PPI with in vitro anticancer potency.Additionally, we introduced several structural modications to optimize the anticancer potential and overall properties of these hits and experimentally conrmed their mode of action as Hsp90-Cdc37 PPI inhibitors.

Virtual screening
To identify new potential inhibitors of the Hsp90-Cdc37 PPI, we conducted a ligand-based virtual screening using a library of DDO-5936 analogs [45][46][47] as the starting point.Four of the most potent Hsp90-Cdc37 PPI inhibitors (Table S1 †), with K d values ranging from 0.50 mM to 5.50 mM, 47 were used as a training set to build a ligand-based pharmacophore model (LBPM) in Fig. 1 Representative natural products and celastrol derivative 41, withaferin A, FW-04-806 and rationally designed small molecules DCZ3112 and DDO-5936, which prevent the formation of the Hsp90-Cdc37 PPI.Blocking the interaction between Hsp90 (PDB ID: 8EOB) and Cdc37 (PDB ID: 2K5B) prevents the loading of Cdc37 clients such as Cdk4 (PDB ID: 2W96) to Hsp90 and thus the formation of the Hsp90-Cdk4-Cdc37 (PDB ID: 5FWP) complex.
6][47] The model was highly selective and retrieved only six active compounds.Since the most potent compounds contained the basic piperazine moiety, neutral analogs like DDO-5936 were not identied as hits in the virtual screening.However, when we used this model in the virtual screening of the library of commercially available compounds, no hits were identied.To improve the hit rate, the initial LBPM was simplied by omitting selected hydrophobic features, hydrogen bond donors and acceptors (Fig. 2b).This decreased its speci-city, as it retrieved three inactive compounds in addition to the six actives.When the simplied LBPM was used in the virtual screening, three structurally similar hits 1-3 were identied (Table 1 and Fig. 2c).Three compounds presented in Table 1 (1-3) were evaluated for their anticancer activity in MCF-7 breast cancer cells, and all three inhibited cell growth within the middle micromolar range (IC 50 = 30-50 mM).Known Hsp90-Cdc37 PPI inhibitor DDO-5936 was used as a control but it did not inhibit the growth of MCF-7 cells with IC 50 value under 50 mM (17% growth inhibition at 50 mM).Among the virtual screening hits, compound 1 demonstrated slightly higher anticancer activity than the other two.Therefore, its affinity for Hsp90 was evaluated using microscale thermophoresis (MST).Due to poor solubility of 1, its binding could only be conrmed qualitatively, as an increase in binding could be detected in concentrations above 39 mM (Fig. S1 †), but the highest concentration without compoundinduced protein aggregation (125 mM) was insufficient to reliably determine its K d value.This conrmation of binding to Hsp90b, along with the antiproliferative activity of compound 1 in MCF-7 breast cancer cells, provided us with a starting point for further hit optimization.

Design, synthesis and evaluation of library A
In the design of library A, modications of the piperidine moiety (colored in blue) and the linker bound phenyl ring (colored in yellow) were planned (Fig. 3).Since the cyanomethyl substituent at the piperidine ring of 1-3 seemed not to be important according to the ligand-based pharmacophore model, it was removed.Similarly, the C-C bond in compound 1 connecting piperidine and pyrimidine rings was replaced with a synthetically more accessible C-N bond.6][47] On the other side of the molecule, the unsubstituted phenyl ring of compound 1 was retained, while linker variations were planned to reduce exibility and investigate potential effects on antiproliferative activity.Apart from removing the methyl group on the pyrimidine of compound 1, the core 2-((pyrimidin-4-yl)amino)thiazole was kept unchanged.
The synthesis of library A (Scheme 1) began with an amide coupling between respective amines and 2-aminothiazole-4carboxylic acid using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt) to produce compounds 4a-c.The resulting 2-aminothiazolamides were reacted with 4,6-dichloropyrimidine in a nucleophilic aromatic substitution to yield compounds 5a-c.To enable the reaction of nucleophilic aromatic substitution, the nucleophilicity of the slightly acidic amino group of 2-aminothiazoles 5a-c was increased by in situ deprotonation using sodium hydride.Nucleophilic aromatic substitution was then performed again using pyrrolidine to give nal products 6a-c and N-Bocpiperazine to produce intermediates 7a-c.In the nal step, the Boc protecting groups of 7a-c were removed by acidolysis to synthesize the nal compounds 8a-c.
Compounds 6a-c and 8a-c were evaluated for their inhibitory effect on the MCF-7 breast cancer cell line (Table 2).The rst set of changes resulted in a slight activity increase from 29.5 ± 5.4 mM for compound 1 to 15.2 ± 0.7 mM and 17.1 ± 1.4 mM for compounds 6b and 8a, respectively.The results also indicate that a basic center is not necessary for the antiproliferative effect in MCF-7 cell line, as both piperazine and pyrrolidine substituents show similar activities.This is in line with previous ndings regarding the SAR of compounds used to set up the pharmacophore model. 46,47The removal of the cyanomethyl substituent, the replacement of the C-C bond with its C-N counterpart, and the changes to the linker between the core thiazole and benzene ring were well tolerated.However, no signicant and consistent improvement in MCF-7 cell growth inhibition was observed for library A, regardless of the introduced changes.Therefore, we decided to replace the aminothiazole core with meta-and para-substituted benzene rings in libraries B and C, respectively.The use of 3-and 4-aminophenyl backbones in libraries B and C enabled further exploration of the chemical space around the central core (Fig. 2).3-and 4-aminophenyl rings comparably afford a slightly different but still similar orientation at the core to that of 2-aminothiazole ring in library A. Additionally, the benzene core provided an alternative synthetic route without the need for a strong base for the activation of 2-aminothiazole, due to the greater inert nucleophilicity of the aniline.Therefore, non-methylated phenethylamine and 2-amino-1phenoxyethane were added to the linker-to-benzene library used in amide bond formation to complement the initial three fragments used in library A. All ve amines were attached to both meta-(library B) and para-aminobenzoic acid (library C) to enable a better comparison of the results.
In the synthesis of libraries B and C (Scheme 2), the respective amides 9a-j were rst prepared by EDC-and HOBtpromoted amide coupling.In the two consecutive nucleophilic aromatic substitutions, 9a-j were rst reacted with 2,4dichloropyrimidine to give intermediates 10a-j.Then, either pyrrolidine or N-Boc-piperazine were attached to yield nal products 11a-j or intermediates 12a-j, respectively.This was followed by Boc-deprotection using TFA-catalyzed acidolysis to complete the synthesis of libraries B and C with nal compounds 13a-j.
Upon preparation, compounds 11a-j and 13a-j were tested for their antiproliferative activities on MCF-7 cells.Unfortunately, the compounds in libraries B (Table 3) and C (Table 4) did not show signicant improvement in cancer cell growth inhibition.The most potent compounds, 13h and 11b, closely followed by 13f, 11i, 11h, and 13g, exhibited similar potency to that of 6b and 8a from library A, with IC 50 values approaching the low micromolar range.Overall, the mean IC 50 values for active compounds in libraries B and C are 27.6 mM and 21.0 mM, Paper RSC Advances respectively, indicating a trend that favors para substitution, though not statistically signicant (p value = 0.087, Kolmogorov-Smirnov t-test).However, the para-substituted set includes three inactive compounds compared to only two in its meta-substituted counterpart.The piperazine substituent, more related to hit compounds, is slightly better tolerated with para substitution.Additionally, the introduction of non-methylated amides conrmed that methyl is not crucial for the antiproliferative effect of the compounds.Considering all ve linkers introduced in libraries A, B, and C, the changes in activity appear to be sporadic, and no denitive conclusion regarding individual fragment combinations can be drawn.

Design, synthesis and evaluation of library D
The last set of changes focused on the pyrrolidine/piperidine part of the molecules, as a nal attempt to optimize their antiproliferative effects.The comparison between compounds containing the piperazine and those with pyrrolidine in libraries A-C revealed that a functional group enabling ionic interaction or hydrogen bonding is not essential, with any of the central cores.To further investigate the effect of substituents at this position, piperidine, morpholine, 4-hydroxypiperidine, and 4-aminopiperidine were attached to 10d and 10g to prepare analogs 14-16, 18-21 and 23-24.In all cases, nucleophilic aromatic substitution was performed for the synthesis of compounds 14-17 and 19-22 (Scheme 3).Boc protecting group was cleaved by acidolysis to give 18 and 23.Additionally, electrophilic bromoacetonitrile was reacted with amine 13g in a nucleophilic substitution to produce compound 24, a close analog of 1, bearing the cyano group.Compounds of library D were then evaluated for their inhibitory potency in the MCF-7 cell line (Table 5).Surprisingly, only 15 and 16 inhibited cancer cell growth with IC 50 values lower than 50 mM, while the other compounds were inactive.
The effects were only visible with the meta-substituted core phenyl ring, suggesting that this core allows a more favorable positioning of the potential hydrogen bond donors and acceptors, as seen in compounds with morpholine (15) and 4hydroxypiperidine (16).Interestingly, compound 18 carrying a 4-aminopiperidine at the same position and the other analogs were not active.Overall, the inactivity of most library D analogs showed that the initial pyrrolidine and piperazine substituents were more suitable for this position.Also, the re-introduction of the cyanomethyl substituent to 13g (19.3 ± 2.0 mM) in compound 24 resulted in a loss of activity.
Initial hit compound 1 and its representative analogs 6c, 8c, 11b, 11g, 13b, and 13g were selected for further evaluation of antiproliferative activity in the Ewing sarcoma (EwS) cell line SK-N-MC and the monocytic leukemia cell line THP-1 (Table 6).The results in EwS cell line were consistent with the MCF-7 data, as 11g was the only inactive compound, and the IC 50 values for 1, 11b, 13b, and 13g were similar in both cell lines.In the remaining 6c and 8c pair, 6c was found to be less potent in EwS, while 8c was a stronger growth inhibitor of SK-N-MC cells than MCF-7 cells.Interestingly, only 8c and 13g retained some inhibitory activity in the leukemia cell line THP-1, while the IC 50 values for the remaining compounds were greater than 50 mM.Surprisingly, the purchased DDO-5936 did not inhibit the growth of any of the tested cell lines with IC 50 values lower than 50 mM.Due to the potency of 8c, the ability of both 8c and 13g to induce apoptosis in the EwS cells SK-N-MC was further evaluated by annexin V and Sytox Blue staining.
Cells were exposed to compounds 8c and 13g at concentrations of 10 mM and 20 mM for 24 and 48 hours (Fig. 4).Both compounds induced apoptosis in SK-N-MC cancer cells.Similar to known inhibitors of Hsp90-Cdc37 PPI FW-04-806 and celastrol that induced apoptosis in breast 37 and pancreatic 50 cancer cells, respectively, an increase in apoptotic cell portion was observed with compounds 8c and 13g.Compound 8c signicantly induced apoptosis at both concentrations and time-points.The proportion of apoptotic cells when exposed to 8c at 20 mM was extensive already aer 24 hours (87.5% vs. 11.4% for the vehicle control) and remained high aer 48 hours treatment (87.7% vs. 9.5% for the vehicle control).Aer treatment with 13g at 20 mM, the proportion of apoptotic cells was 42.8% and 56.8% aer 24 hours and 48 hours, respectively.When treated with 10 mM compound 13g, cells initially underwent apoptotic cell death (27% of cells); however, they appeared to recover aer 48 hours (proportion of apoptotic cells 11.4%), and the proportion of apoptotic cells here was not signicantly different from the vehicle control.Based on these results, compound 8c is more efficient in inducing cell death.These ndings are consistent with results from the proliferation assay, where 8c exhibited higher cytotoxicity compared to 13g in SK-N-MC cells.

Investigation of the mechanism of action
Compounds 8c and 13g were selected to examine the mode of action of the prepared compounds, and their binding to fulllength Hsp90b was studied using microscale thermophoresis (MST).Both compounds bound to Hsp90b with an affinity in the micromolar range, with K d values of 70.8 ± 11.0 mM for 8c and 73.3 ± 2.0 mM for 13g (Fig. 5, S2A and S3A †).The binding affinity of 8c for Hsp90b was further investigated in the presence of increasing concentrations of the full-length Cdc37.In three consecutive experiments, the concentration of Cdc37 was increased from the initial 0.5 mM to 1.0 mM and nally to 2.0 mM, reaching the reported K d range of Cdc37 for Hsp90. 45,51The addition of Cdc37 resulted in a concentration-dependent decrease in the affinity of 8c for Hsp90b, with K d values increasing from 70.8 mM in the absence of Cdc37 to 81.7 mM (0.5 mM Cdc37), 90.2 mM (1 mM Cdc37), and 98.2 mM (2 mM Cdc37) (Fig. 5 and S3B-D †).This indicated that 8c binds to Hsp90b in the area of the Hsp90-Cdc37 interaction interface, thereby disrupting their PPI. 52Additionally, when the affinity of 13g for Hsp90b was tested in the presence of 1.0 mM Cdc37, the binding affinity within the applied concentration range was too low for quantication (Fig. S2B †).These results suggest that compounds 8c and 13g are displaced from Hsp90b by Cdc37, indicating their potential to disrupt the Hsp90-Cdc37 interaction.
In the absence of the experimentally determined binding site of these Hsp90-Cdc37 inhibitors, the binding of 13g to the fulllength Hsp90b was explored by saturation transfer difference (STD) NMR spectroscopy under quantitative conditions.The STD amplication factors (Fig. 6) indicate that the aromatic regions of 3-(phenylamino)pyrimidine and peripheral phenyl ring of 13g are in the closest proximity with the Hsp90b binding site.Meanwhile, the contributions of the aliphatic protons in the linker between the two phenyl rings were slightly weaker.Similarly, the piperazine ring forms weaker contacts with the protein than the aromatic parts, but this part of the inhibitor also plays an important role according to the SAR of library D.
Binding of inhibitors 13g to Hsp90b was further conrmed by transferred NOESY (trNOESY) experiments, as negative NOEs with the same sign as the diagonal peaks were observed between the protons of inhibitor in the presence of Hsp90b (Fig. 7a).The NOEs were observed only between adjacent molecular segments, suggesting that 13g adopts an extended conformation when bound to Hsp90b.The calculated conformation of 13g using the distance constraints from trNOESY spectrum is presented in Fig. 7b.The effect of Hsp90-Cdc37 PPI inhibitors 8c, 13g, and DDO-5936 on Hsp90 client protein levels was investigated in MCF-7 cells.As shown in Fig. 8, both 8c and 13g decreased the concentration of kinase clients Cdk4, c-Raf, IGF1R, and Akt at 50 mM, with varying statistical signicance.The effect on client proteins was more pronounced for compound 8c, with c-Raf and Cdk4 being the most affected.Surprisingly, the decrease was most pronounced for ERa, as it was the only Hsp90 client protein signicantly inuenced even at the lower 20 mM concentration of 8c and 13g.ERa was also the only client protein that was downregulated by DDO-5936 with statistical signicance.This was unexpected, as Cdc37 is primarily reported to be important for the kinome-related part of the Hsp90 clientele. 28,32However, the androgen receptor 53 and guanylyl cyclase receptor 48 have also been shown to rely on Cdc37 for recruitment to Hsp90, although they are not protein kinases.Additionally, Hsp70 and Hsp90 levels were examined to assess the heat shock response.Importantly, no increase in either heat shock protein was observed.On the contrary, a statistically insignicant decrease in Hsp70 levels was noted, especially at 50 mM of 8c, indicating another potential benet of inhibiting Hsp90-Cdc37 PPI over classical N-terminal Hsp90 inhibition. 34

Conclusion
In the present work, we have demonstrated the viability of using ligand-based virtual screening to design new inhibitors of the Hsp90-Cdc37 PPI, and thus demonstrated that the current pool of known inhibitors enables this design approach.Our initial hit compounds exhibited anticancer potency, which we further optimized in our SAR studies.However, as observed in previous studies, rmly establishing SAR has been challenging.Additional exploration of the exact binding site of these inhibitors at the Hsp90-Cdc37 interface using full-length proteins would  greatly benet the research community.We have demonstrated that compounds 8c and 13g both bind to Hsp90 in the midmicromolar range, interfere with the Hsp90-Cdc37 PPI, and exhibit in vitro anticancer activity against Ewing sarcoma, breast cancer, and leukemia cell lines.Additionally, they effectively decrease the levels of known Hsp90-Cdc37 client proteins, such as protein kinases Cdk4 and c-Raf.Importantly, compounds 8c and 13g do not induce a heat shock response, a known obstacle for N-terminal Hsp90 inhibitors in their clinical development.This absence of HSR makes the Hsp90-Cdc37 PPI and its inhibitors clinically signicant and worthy of further investigation.

Library preparation
A library of 556 000 compounds from commercial providers was prepared based on the diversity sets from Enamine, Asinex, ChemBridge, Maybridge, LifeChemicals, Vitas-M and Key-Organics.Libraries were downloaded in SDF format, merged and duplicates removed using the LigandScout Database Merger and Duplicates Remover nodes as implemented in the Inte : Ligand Expert KNIME Extensions.
The 14 most potent Hsp90-Cdc37 PPI inhibitors, with K d values ranging from 0.50 mM to 20 mM, were used as a training set of active compounds for building a ligand-based pharmacophore model. 49][47] For each library, a maximum of 200 conformations were generated for each molecule using LigandScout's iCon algorithm with the default "BEST" settings [max.number of conformers per molecules: 200, timeout (s): 600, RMS threshold: 0.8, energy window: 20.0, max.pool size: 4000, max.fragment build time : 30].Each library was saved in LDB (LigandScout database format) using LigandScout's idbgen algorithm with default settings (write all properties and remove duplicates).

Ligand-based pharmacophore modeling
Four most potent Hsp90-Cdc37 PPI inhibitors 47 were used for creation of ten ligand-based pharmacophore models in LigandScout 4.4 Expert.The models were generated using the following ligand-based pharmacophore creation settings: scoring function: pharmacophore t and atom overlap; pharmacophore type: merged feature pharmacophore; number of omitted features for merged pharmacophore: 4; partially matching features optional, threshold (%): 10.0; feature tolerance scale factor: 1.0; maximum number of result

Virtual screening
The selected ligand-based pharmacophore model was used to query the library of 556 000 commercially available diverse compounds, prepared as described using LigandScout 4.4 Expert.The settings included: scoring function: pharmacophore-t, screening mode: match all query features; retrieval mode: stop aer rst matching conformation; max.number of omitted features: 0 and check exclusion volumes: true.Virtual hits were ranked according to pharmacophore t score.Three virtual screening hits LAS 58000795 (1), LAS 58000810 (2) and LAS 58000778 (3) were purchased from Asinex.

Synthesis and characterization
To perform the synthesis reagents and solvents used were acquired from various vendors like Sigma-Aldrich (St. Louis, MO, USA), Enamine Ltd (Kyiv, Ukraine), and Fluorochem Ltd (Derbyshire, UK), and were used without any further purication.To determine the retention factors analytical thin-layer chromatography silica gel aluminum sheets were used (0.20 mm; 60 F254; Merck, Darmstadt, Germany).To purify the compounds with ash column chromatography silica gel 60 (particle size, 230-400 mesh) was used as the stationary phase.
For recording the NMR spectra ( 1 H and 13 C) to analyte the chemical compounds 400 MHz NMR spectrometer (Bruker Advance 3, Bruker, Billerica, MA, USA) was used.The splitting pattern designation was as follows: s, singlet; d, doublet; dd, double doublet; t, triplet; dt, double triplet; ddd, double of doublet of doublet; q, quartet; and m, multiplet.For the monitoring of purity of the nal products ultra-high performance liquid chromatography (UHPLC) (Thermo Scientic Dionex UltiMate 3000 UHPLC modular system, Thermo Fischer Scientic Inc., Waltham, MA, USA) was used, which was equipped with a wavelength detector to perform the purity determination at 254 nm.The method used for purity determination was set up as follows: C 18 column (Waters Acquity UPLC® HSS C18 SB column, 2.1 mm × 50 mm, 1.8 mm); temperature: 40 °C; injection volume 5 mL; ow rate: 0.3 mL min −1 .The mobile phase consisted of varying portions of Solvent A (0.1% TFA in H 2 O) and Solvent B (MeCN), using the gradient as follows: 0 / 7 min 5% B; 7 / 8 min 95% B. Mass spectra were recorded using expression CMS L mass spectrometer (Advion Inc., Ithaca, NY, USA), while to obtain highresolution mass spectra Exactive Plus Orbitrap mass spectrometer (Thermo Scientic Inc., Waltham, MA, USA) was used.DDO-5936 was purchased from MedChemExpress.

General procedure A
The corresponding carboxylic acid (1 equiv.) was dissolved in N,N-dimethylformamide (DMF) (20 mL).The solution was put on an ice bath and EDC (1.3 equiv.),HOBt (1.3 equiv.)and Nmethylmorpholine (NMM, 2 equiv.)were added.The reaction was stirred at 0 °C for 20 minutes.The corresponding amine (1 equiv.) was then added, and the reaction mixture was stirred overnight at room temperature.The solvent was evaporated, and the residue was taken up in EtOAc (100 mL) and NaHCO 3 (50 mL).The layers were separated, and the organic layer was further washed with NaHCO 3 (2 × 50 mL) and 1% citric acid (3 × 30 mL).The organic layer was washed with brine (40 mL), then dried over Na 2 SO 4 , ltered and the solvent evaporated under reduced pressure.

General procedure B
The corresponding 4-substituted 2-aminothiazole (1 equiv.)and 4,6-dichloropyrimidine (1 equiv.)were dissolved in dry THF (20  mL).Then 60% NaH on mineral oil (2.5 equiv.) was added on an ice bath.The temperature was gradually increased to ambient temperature and then the reaction mixture stirred for 3 hours at room temperature.Then, saturated NH 4 Cl (aq) (40 mL) was added, and the mixture was concentrated under reduced pressure.EtOAc (50 mL) was added to the residue and the layers were separated.The aqueous layer was additionally extracted with EtOAc (50 mL).The combined organic phases were washed with brine (30 mL), dried over Na 2 SO 4 , ltered, and then the solvent evaporated under reduced pressure.The products were additionally puried by ash column chromatography.

General procedure C
The corresponding 6-chloro-N-substitutedpyrimidin-4-amine (1 equiv.)and respective amines (1 equiv.)were dissolved in DMF (10 mL).In the case of pyrrolidine (7 equiv.), the reaction mixture was stirred at 70 °C overnight.In the case of N-Bocpiperazine, piperidine, morpholine, 4-hydroxypiperidine and 4-(N-Boc-amino)piperidine (5 equiv.),Et 3 N (2 equiv.) was also added to the reaction mixture, which was then heated to 80 °C and le to stir over three days for 4-(N-Boc-amino)piperidine and N-Boc-piperazine and overnight for pyrrolidine, morpholine and 4-hydroxypiperidine.Next, the volatiles were evaporated, and the residue was taken up in ethyl acetate (30 mL) and washed with 1% citric acid (3 × 20 mL), brine (20 mL), dried over Na 2 SO 4 , ltered, and the solvent evaporated under reduced pressure.When necessary, the crude products were additionally puried by ash column chromatography.

General procedure D
The Boc-protected amine (1 equiv.) was dissolved in DCM (10 mL) and TFA (20 equiv.) was added.The reaction mixture was stirred overnight at room temperature.The solvent was evaporated and the product was puried by ash column chromatography.

General procedure E
The meta-or para-substituted aminobenzamides (1 equiv.)and 4,6-dichloropyrimidine (1.5 equiv.)were dissolved in DMF (30 mL).Then, DIPEA (2 equiv.) was added, and the reaction mixture was heated to 100 °C and le to stir for 3 days.The solvent was evaporated under reduced pressure and the residue was taken up in EtOAc (50 mL) and water (50 mL).The layers were separated, and organic layer was additionally washed with water (50 mL), brine (30 mL), dried over Na 2 SO 4 , ltered, and the solvent evaporated under reduced pressure.The product was additionally puried by ash column chromatography.

Cell viability assay
The antiproliferative activity of the compounds was evaluated against MCF-7 (ATCC-HTB-22; ATCC), SK-N-MC (a kind gi from Beat Schäfer) and THP-1 (ATCC-TIB-202; ATCC) cancer cell lines using the MTS assay (Promega, Madison, WI, USA).Cells were seeded in 96-well plates at a density of 2 × 10 4 cells per mL in 100 mL of growth medium for THP-1 and were treated with compounds and controls directly, while 3 × 10 3 cells per mL for MCF-7 and SK-N-MC were seeded in 100 mL of growth medium and allowed to adhere overnight following the treatment.In all cases the cells were treated with respective newly prepared compounds, positive controls (17-DMAG for MCF-7 and SK-N-MC; PU-H71 for THP-1) and a vehicle control (0.5% DMSO).The incubation time aer treatment was 72 hours, then Cell-Titer96 ® Aqueous One Solution Reagent (Promega, Madison, WI, USA) was added to each well.Following a 3 hours incubation, absorbance was measured at 492 nm using BioTek's Syn-ergy™ 4 Hybrid Microplate Reader (Winooski, VT, USA).The measurements were performed in two biological repetitions and each time the experiment was performed in a triplicate.The IC 50 values, representing the concentration of inhibitor required for half-maximal inhibition, are the average of three independent experiments and were calculated using GraphPad Prism 9.5.0 soware (San Diego, CA, USA).

Apoptosis assay
Apoptosis of SK-N-MC cells was evaluated by detecting phosphatidylserines using R-phycoerythrin-annexin V conjugate (R-PE annexin V; Invitrogen, Carlsbad, CA, USA) and nucleic acids in dead cells using SytoxBlue Dead Cell Stain (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions.Briey, 2.5 × 10 5 SK-N-MC cells were seeded in six-well plates.Aer 24 hours, cells were washed with PBS and treated with 8c and 13g in 10 and 20 mM concentrations for 24 and 48 hours.The medium with detached cells was then collected, and attached cells were harvested and combined with the detached cells.Following two washing steps with cold PBS, cells were resuspended in 100 mL annexin-binding buffer (Invitrogen, Carlsbad, CA, USA) containing 2.5 mL annexin V-R-PE solution and 750 nM SytoxBlue, and incubated in the dark for 15 minutes at room temperature.Before measurement, 200 mL of annexin-binding buffer was added.A minimum of 10 000 events were collected using a ow cytometer (Attune NxT; Invitrogen, Carlsbad, CA, USA).Annexin V (ANV)-/SYTOX Blue (SB)indicates viable cells, ANV+/SB− indicates early apoptotic cells, ANV+/SB+ indicates late apoptotic cells, and ANV−/SB+ indicates necrotic cells.

Western blot
MCF-7 cells were treated with compounds 8c (20 mM and 50 mM), 13g (20 mM and 50 mM) and DDO-5936 (20 mM) or 0.5% DMSO as a negative control.The cells were incubated with the compounds or negative control for 24 hours.Aer incubation, the cells were rinsed with PBS (Gibco, Thermo Fisher Scientic, Waltham, MA, USA) and lysed using RIPA buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 1 mM EDTA.The RIPA buffer was further supplemented with Halt™ Protease Inhibitor Cocktail and Halt™ Phosphatase Inhibitor Cocktail (both from Thermo Fisher Scientic, Waltham, MA, USA) at a 1 : 100 dilution.The lysates were then frozen for at least 24 hours.Upon thawing, the lysates were sonicated and centrifuged at 15 000 rpm for 20 minutes at 4 °C.Only the supernatants were collected, and protein concentration was measured using the DC protein assay (Bio-Rad, Hercules, California, USA).Proteins (20 mg) were separated by SDS-PAGE on a 10% acrylamide/bisacrylamide gel, running at 80 V for 15 minutes followed by 130 V for 60 minutes.The separated proteins were then transferred onto a nitrocellulose membrane using the iBlot 2 Dry Blotting System (Thermo Fisher Scientic, Waltham, MA, USA).The membranes were blocked with 5% BSA for 1 hour at room temperature to prevent nonspecic binding.Primary antibodies were then added, and the membranes were incubated overnight at 4 °C.The primary antibodies (Cell Signaling, Danvers, MA, USA) used included anti-b-ActinMouse mAb (1 : 5000), anti-Hsp90 Rabbit mAb (1 : 1000), anti-Hsp70 Mouse mAb (1 : 1000), anti-IGF1R Rabbit (1 : 1000), anti-CDK4 Rabbit (1 : 1000), anti-ERa Mouse mAb (1 : 1000), anti-Akt Rabbit mAb (1 : 1000), and anti-c-Raf Rabbit mAb (1 : 1000).Secondary antibodies included antirabbit IgG, HRP-linked antibody (1 : 10 000), and anti-mouse IgG, HRP-linked antibody (1 : 10 000), which were incubated with the membranes for 1 hour at room temperature.Following washes, SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientic, Waltham, MA, USA) was applied.Visualization of the blots was performed using the UVITEC Cambridge Imaging System (UVITEC, Cambridge, UK), and densitometric analysis of the western blot bands was conducted using NineAlliance soware.Relative protein densities were calculated in relation to b-actin, used as a loading control.

Expression and purication of the full-length Hsp90b
For the protein expression of full-length Hsp90b, we received plasmids as a kind gi from Dr Asta Zubriené at the Institute of Biotechnology, Vilnius University, Lithuania.Hsp90b, tagged with N-terminal 6×His-tag, was expressed in Escherichia coli strain Rosetta.The cells were cultured in LB media supplemented with kanamycin (50 mg mL −1 ) and chloramphenicol (34 mg mL −1 ) at 37 °C, and protein expression was initiated with 0.5 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) when the OD 600 reached 0.8.Following induction, the cells were incubated for 16 hours at 18 °C before being harvested by centrifugation.The cells were resuspended in a lysis buffer containing 40 mM potassium phosphate pH 8.0, 400 mM KCl, 10 mM imidazole, and protease inhibitors (Sigma), and then lysed by sonication.Aer centrifugation, the proteins were initially puried using a Ni 2+ -affinity HisTrap column (GE Healthcare).Impurities were washed away with lysis buffer containing 20-40 mM imidazole, and Hsp90b was subsequently eluted with lysis buffer containing 300 mM imidazole.The purication process continued with size exclusion chromatography (SEC) using a Superdex-200 (16/600) column (GE Healthcare) and a running buffer of 50 mM Tris pH 7.5 at room temperature, 300 mM KCl.The purity of the fractions was conrmed by SDS-PAGE, and they were then concentrated.For the NMR experiments, Hsp90b was dialyzed against NMR buffer (50 mM potassium phosphate pD 7.5, 100 mM KCl, 2 mM DTT (98%, D10) in D 2 O) and subsequently frozen in liquid nitrogen.

Microscale thermophoresis
The full-length Hsp90b was labeled using the Monolith His-Tag Labeling Kit RED-tris-NTA following the manufacturer's instructions (NanoTemper Technologies GmbH, Munich, Germany).The proteins were rst diluted to a concentration of 8 nM in assay buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 5% ethanol, and 10 mM MgCl 2 ).To determine the K d values, the proteins were mixed with the test compounds in a 1 : 1 ratio as stated in the manufacturers protocol.The nal concentration of Hsp90b was 4 nM, with compounds added in the following ranges: 1000 mM to 5.86 mM for 8c; 312.5 mM to 0.15 mM for 13g and 125 mM to 3.91 mM for 1.Two independent K d determinations were performed for compounds 8c and 13g.
Higher compound concentrations both with and without Cdc37 resulted in aggregation due to insufficient solubility, so these measurements were excluded (MST curves colored in grey).
Next, Cdc37 (AR09008PUN, OriGene Technologies, Inc., Rockville, MD, USA) was acquired for the purpose of PPI inhibition evaluation between full-length Hsp90b and Cdc37.The K d values of compounds 8c and 13g with Hsp90b (4 nM) were reevaluated in the presence of Cdc37 (0.5 mM, 1 mM and 2 mM for 8c and 1 mM for 13g) using the following concentration ranges of inhibitors: 500 mM to 5.86 mM for 8c and 312.5 mM to 1.95 mM for 13g.In all experiments the compounds were incubated with the proteins for 15 minutes in the dark at room temperature.The mixtures were then loaded into Monolith NT.115 Premium Capillaries (NanoTemper Technologies GmbH, Munich, Germany).Thermophoresis was induced at 1475 ± 15 nm and measured using a Monolith NT.115 pico instrument (Nano-Temper Technologies GmbH, Munich, Germany).Measurements were conducted at ambient temperature (24-25 °C), with excitation power set to 20% and MST power set to 40%, and a laser on time of 5 seconds.To calculate the K d values, the average uorescence responses for each concentration were plotted against the logarithm of compound concentrate on using GraphPad Prism soware (GraphPad Soware, Inc., La Jolla, CA).

Ligand-observed protein NMR studies
High-resolution NMR spectra of 13g were recorded using a Bruker Avance Neo 600 MHz spectrometer with a cryoprobe at 25 °C.Data collection employed pulse sequences from the Bruker library, and analysis was conducted using Bruker Topspin 4.2.0.The residual water signal was suppressed by excitation sculpting 54 with a 5 ms selective pulse, and a T 1 r lter of 100 ms was used to remove background protein resonances.
The 1 H spectral widths were set at 5263 Hz.NMR samples were prepared in a buffer containing 50 mM potassium phosphate (pD 7.5), 100 mM KCl in D 2 O, 5 mM MgSO 4 , 2 mM DTT-d 10 , 0.02% NaN 3 , and 2% DMSO-d 6 .Full proton assignment (see ESI Fig. S6 and Table S2 †) was achieved using TOCSY, NOESY, ROESY, and HSQC spectra.The 1 H STD spectra were recorded with a protein : ligand ratio of 1 : 200, with protein and ligand concentrations of 1.5 mM and 3 mM.For the 1 H STD ligand epitope mapping experiments, 55 65 536 data points (6.23 s and acquisition time) were used, with a relaxation delay of 1.63 s and 5120 scans.A short protein saturation time of 0.5 s was utilized to minimize the effect of relaxation on the STD amplication factors. 56Selective onresonance saturation of Hsp90b was performed at −0.827 ppm, with the transmitter offset referenced to 4.70 ppm.Off-resonance irradiation for the reference spectrum was applied at 30 ppm.The spectra were zero-lled and apodized using an exponential line-broadening function of 3 Hz.Errors in the STD amplication factor were estimated using the spec-ied formula: 57  S3 and  S6 †) for all protons are below 4%.The trNOESY 58 spectra were recorded with 4096 data points in t 2 , 64 scans, 360 complex points in t 1 , and a relaxation delay of 1.5 s.A mixing time of 150 ms was selected according to the binding affinity of the derivatives to compromise between sufficient signal-to-noise ratio and reduced spin diffusion.The spectra were apodized with a squared sine bell function shied by p/2 in both dimensions.Distances for 13g were calculated from the cross-peak volumes, assuming the integrated intensity of proton pairs 2 0 , 4 0 and 1 0 , 5 0 in the para-substituted phenyl ring with a distance of 2.5 Å and that the distances of the protons in pyrimidine ring are the same.

Conformation of compound 8c and 13g from NMR experiments
Conformation of 13g were calculated in Schrodinger Suite using distance constraints from trNOESY experiments (Table S4 †).For conformational search systematic torsional sampling method with default settings was used.

Fig. 2
Fig. 2 Overlay of the optimized DDO-5936 analog 18 h with (a) initial and (b) simplified ligand-based pharmacophore model.(c) Overlay of the virtual screening hit 1 with the simplified ligand-based pharmacophore model.Pharmacophore features are presented as follows: hydrogen bond acceptorred sphere, hydrogen bond donorgreen arrow or sphere, hydrophobic featureyellow sphere, aromatic ringblue disc, positively ionizableblue star.

Fig. 3
Fig.3Schematic representation displaying our work plan following the discovery of hit compound 1.First, library A was designed, and the results of the biological evaluation steered the planning of libraries B and C, while library D was designed at the very end.The optimisation was aimed at the improvement of in vitro anticancer effects of the compounds.

Fig. 4
Fig. 4 Induction of apoptosis in SK-N-MC cells by compounds 8c and 13g.SK-N-MC cells were exposed to 10 mM or 20 mM 8c or 13g.After 24 h or 48 h, apoptosis was measured by annexin V-PE/SytoxBlue staining.Percentages apoptotic cellsleft, and representative scatter plots of annexin V-PE (x-axis) and Sytox blue (y-axis)right.Data in the bar charts are presented as the means ± SD of at least three biological replicates.

Fig. 5
Fig. 5 Schematic representation of binding of 8c and 13g to Hsp90 and PPI inhibition investigation for 8c.

Fig. 6
Fig. 6 1D 1 H STD NMR spectra for the compound 13g recorded at an Hsp90b : ligand ratio of 1 : 200 and 600 MHz.The molecular structure illustrates the proton nomenclature and the color-coded relative degrees of saturation of the individual protons.The STD amplification factors were normalized to the intensity of the signal with the largest STD effect.Reference STD spectra (top) with proton assignment and difference STD spectra (bottom) are shown.The unassigned proton signals between 3.5 and 3.8 ppm belong to the protein buffer with glycerol.The proton signals were calibrated to the DSS signal at 0.0 ppm.The spectra are not to scale.

Fig. 7
Fig. 7 (a) The trNOESY spectrum of 13g in the presence of Hsp90b with the molecular structure illustrating the atom nomenclature and the NOE connectivities between the protons of the different molecular segments.The NOE connectivities of the magnetically equivalent protons with overlapping signals are shown schematically for one orientation of the corresponding rings only.(b) Calculated conformation of 13g based on distance constraints from trNOESY experiment (Table S4 †).

2 N
STD and N REF are noise levels in STD and reference spectra.I STD and I REF are signal intensities in STD and reference spectra.The relative errors of the STD amplication factors (Tables

Table 3
Antiproliferative IC 50 values (mean ± SD) of the compounds 11a-e and 13a-e of library B determined in MCF-7 breast cancer cell line © 2024 The Author(s).Published by the Royal Society of Chemistry RSC Adv., 2024, 14, 28347-28375 | 28353

Table 4
Antiproliferative IC 50 values (mean ± SD) of the compounds 11f-j and 13f-j of library C determined in MCF-7 breast cancer cell line